The pathophysiological mechanisms leading to anxiety and depression are not understood. The prevalence of these disorders is increasing, imposing a significant burden on our health care. Accumulating data suggest that gut microbiota affects the function of the brain and its chemistry. It is likely that multiple mechanisms are involved, including bacterial production of neuroactive molecules or activation of immune pathways. We will seek to study the mechanisms, by which gut bacteria lead to development of anxiety and depression using gnotobiotic murine models. In the R21 phase (Aim 1) we will develop a gnotobiotic model of anxiety and depression by colonizing germ- free mice with stool microbiota from patients with Generalized Anxiety Disorder (GAD) and Major Depressive Disorder (MDD), using samples from our clinical bio bank at McMaster University. We will characterize mouse behavior, microbiota and metabolomic profiles using 16S DNA-based Illumina sequencing and liquid chromatography-mass spectrometry (LC-MS), respectively. As stress has been shown to play a role in depression and anxiety, we will apply a psychosocial stressor to a group of gnotobiotic mice with signs of immune activation. We hypothesize that bacteria communicate with the brain through both neural and immune pathways, which include neuroactive trace amines and inflammatory cytokines, such as IL-1. These specific pathways will be the focus of our research in the R33 phase. Using selected set of microbiota identified in Aim1, we will colonize additional groups of germ free mice and study in detail the underlying mechanisms. The mouse behavior will be correlated with gut, serum and brain neurotransmitters, as well as neuroactive and immunomodulatory metabolites of bacterial origin. We will assess neuronal activation in specific brain areas by immunohistochemistry. We will use pharmacological tools and genetically modified mice to study the exact pathways of microbiota-brain communications. Taking advantage of our novel approach, which enables to culture >92% of human gut bacteria, we will identify and isolate the bacteria associated with anxiety and/or depression-like behavior. To confirm the cause-effect relationship, we will mono or poly-colonize additional groups of germ-free mice with specific bacteria, or group of bacteria linked to anxiety and/or depression, and assess mouse recipient behavior and brain chemistry.